Interpretive Summary: Phytotoxin (Rs-toxin) is produced by the necrotrophic soil-borne fungus Rhizocotnia solani during the infection process. The major bioactive component of the toxin has been unclear. In the present study, an array of extraction methods coupling with ultraviolet (UV)- and visible-wavelength light scanning, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) analyses, were used to identify the major bioactive component of the toxin. The activated charcoal adsorbent method proved to be effective for isolation of Rs-toxin from the culture filtrate of R. solani. A simple and reliable bioassay system, the floating soakage method, was developed to evaluate the phytotoxicity of Rs-toxin. Using Ninhydrin test, Molish’s and Fehling’s reaction the bioactive components of Rs-toxin were determined to be carbohydrates other than amino acids or proteins in the aqueous fraction. The partially purified Rs-toxin was found to contain only six saccharides: ribose, arabinose, xylose, mannose, glucose and galactose as determined by GC/MS. By means of peak area normalization, the relative amounts of these six saccharides were determined to be: 83.63% glucose, 14.3% mannose, 0.83% arabinose, 0.83% xylose, 0.20% ribose and 0.07% galactose. These saccharides were also shown to possess obvious maceration effects on detached rice leaf tissues suggesting that they are the major bioactive component of the toxin of R. solani.

Technical Abstract:
Phytotoxins (Rs-toxins) produced by R. solani are known to play an important role in the pathogenesis of this fungal pathogen, but the principal components of this phytotoxin were quite different from previous studies. To isolate and characterize the bioactive components of the Rs-toxin produced by the rice blight pathogen, R. solani AG-1 IA, various extraction and purification methods, several chemical reactions, ultraviolet (UV)- and visible-wavelength light scanning, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) analyses were used. The maceration effects of Rs-toxin on rice leaf tissues were investigated to verify the bioactivity of Rs-toxin. Through a comparison of four bioassays, a simple and reliable bioassay system, the leaf floating soakage method, was established for evaluating the phytotoxicity of culture filtrate, different extraction fractions and partially purified Rs-toxin. The results of Molish’s reaction, Ninhydrin test and Fehling’s reaction suggested that the bioactive components of Rs-toxin were carbohydrates other than amino acids or proteins. The results of bioassay revealed that no organic solvent was effective for extracting the bioactive components from the culture filtrate of R. solani, and the phytotoxic components were found to be in the aqueous fraction after extraction with six organic solvents. The activated charcoal adsorbent method was used and proved to be effective for isolation of Rs-toxin from the culture filtrate of R. solani confirmed by bioassay. By using GC/MS detection technique, the partially purified Rs-toxin was found to contain six saccharides: ribose, arabinose, xylose, mannose, glucose and galactose. However, no N-acetylgalactosamine or N-acetylglucosamine could be detected in this toxin. By means of peak area normalization, the relative amounts of these six saccharides were: 83.63% for glucose, 14.3% for mannose, 0.83% for arabinose, 0.83% for xylose, 0.20% for ribose and 0.07% for galactose. The result of LC/MS/MS indicated that neither phenylacetic acid nor its derivatives was detected in Rs-toxin. Taken together, it is concluded that Rs-toxin consists of six kinds of saccharides and has obvious maceration effects on rice leaf tissues.